why my Northern blot do not work?

[alex dobrovic]

There has been other people with problems with Hybond N+ so we changed to
Brightstar from
Ambion (no affiliation) on the basis that they specialised in RNA. We have
had good results. It costs more but then so does labour and RNA  and loss
of morale.

.>I did my Nothern blot, but it did not have any signals. I know the gene
>is highly expressed by previous differential screening of the cDNA
>library. I followed the protocal of the Amersham booklet with the Hybond
>N+ membrane. That is what I did: transfer the RNA use 20x ssc, briefly
>wash with 2xssc, UV crosslink, store in -4C for about one week,
>pre-hybidize for 3 days, hybridize for 16 hours, wash with 2xsspe 0.1%
>SDS two times @ r.t. 10min, 1xsspe @ 65C for 15min, 0.1xsspe @ 65C for
>about 45min. After I did the RNA transfer I checked gel, there was no
>EtBr Stained RNA left on the gel. The Southern blot I did in the same
>box for hybridize and wash  worked beautifully. So I really can not
>figure out what is wrong. If any one can help me, I will be very
>grateful, thanks!
>
>Hongyan Wang
>Texas A&M university


Alexander Dobrovic, Ph.D.
Chief Medical Scientist
Department of Haematology-Oncology
The Queen Elizabeth Hospital
Woodville, SA 5011, Australia

Affiliate Senior Lecturer
Department of Medicine
University of Adelaide

Tel 61-8-8222 7676 (new as from April 1998)
Fax 61-8-8222 6046
adobrovi at medicine.adelaide.edu.au